Breast cancer is the most common malignancy in women worldwide and the leading cause of cancer related death. More than 1.2 million cases are diagnosed every year, affecting 10-12% of the female population and accounting for approximately 500,000 deaths per year worldwide. The primary cause of morbidity and mortality in women with breast cancer is metastasis. We currently do not have the technology to identify breast cancer patients with localized and regional disease who are at risk for metastatic disease. As a result, 80% of patients receive aggressive treatment that is necessary for patients with advanced breast cancer, although only about 10 to 15% of patients develop metastasis within 3 years of diagnosis. Given the significant short- and long-term complications of chemo- and radiotherapy, and its impact on quality of life of survivors, development of prognostic markers of metastasis and identification of anti-metastatic targets would greatly benefit women with breast cancer. The prognostic factors in current use include the number of positive lymph nodes, tumor size, grade and receptor status, and Mammaprint and OncotypeDX. Although all of these tools are in use in the management of breast cancer patients, they are derived from molecular sub-typing of whole tumor tissue and give no information about the presence of migratory and disseminating tumor cells within the tumor tissue as well as little insight into the specific mechanisms of metastasis. Hence, it is not clear how to improve their use within personalized treatment plans concerning risk and treatment response of metastasis. To define the mechanisms responsible for tumor cell migration and dissemination to distant sites, we developed multiphoton microscopes for observing and collecting the migratory and metastatic tumor cells from mammary tumors In live mice in real time. Using this technology we observed the importance of tumor cell-macrophage interactions in metastasis and discovered the human invasion signature (HIS), the expression profile of tumor cells during metastatic dissemination. We also identified the sites of tumor cell intravaation used for dissemination in vivo and named them TMEM for the Tumor MicroEnvironment of Metastasis. We hypothesize that tumor cells with increased activation of the pathways in the HIS can assemble TMEM, thereby increasing Intravasation. We have developed methods to measure the activation of the migration, adhesion and invadopod assembly pathways within the HIS and both intravasation and extravasation activities in vitro and in vivo which will allow us to test this hypothesis. RELEVANCE (See instructions): The results of testing the hypothesis that tumor cells with increased activation of the pathways in the HIS can assemble TMEM, thereby increasing intravasation, have a high potential of being translational by application to determine risk of distant recurrence, and to personalize management of breast cancer patients at risk for metastatic disease. PROJECT/PERFORIVIANCE SITE(S) (if additional space is needed, use Project/